Nucleic acid encoding human EP3 prostaglandin receptor

ABSTRACT

A gene encoding the human EP 3  prostaglandin receptor has been cloned and sequenced. The protein encoded by this gene has seven transmembrane domains and is 81% homologous to the murine EP 3  receptor. Two variants that differ in their carboxy terminal coding sequence, and one variant that differs in its 3&#39; untranslated sequence only, have also been cloned. The proteins, when expressed in eukaryotic cells, are capable of binding prostaglandins and their agonists and regulating adenylate cyclase activity in response to prostaglandins.

FIELD OF THE INVENTION

This invention relates to the cloning and expression of the human EP₃prostaglandin receptor. Methods of identifying compounds capable of bothbinding to and activating the human EP₃ receptor are also disclosed.

BACKGROUND OF THE INVENTION

Ptostaglandins are a group of lipid-soluble hormone mediators derivedfrom the metabolism of arachidonic acid via the cyclooxygenase enzymaticpathway. In the prostaglandin biosynthetic pathway, arachidonic acid isfirst converted to the endoperoxide PGH2 by PGH2 synthases followed bythe cell-specific isomerization or reduction of PGH2 to the activeprostaglandins: PGD₂, PGE₂, PGF₂α, prostacyclin (PGI₂) and thromboxane(TxA₂). Following enzymatic conversion, prostaglandins exert theiractions locally on the cells in which they were synthesized (autocrine)and/or on nearby cells (paracrine) through specific G protein-coupledreceptors (Smith, (1992) Am. J. Physiol., 263: F181-F191) to eitherstimulate or inhibit the production of second messengers. Prostaglandinselicit a diverse spectrum of often opposing biological effects includingmuscle contraction and relaxation, platelet aggregation, vasodilationand inflammation.

PGE₂ exhibits a broad range of actions in a number of tissues by bindingto at least three EP receptor subtypes. It acts throughpharmacologically distinct stimulatory (EP₂) and inhibitory (EP₃) PGEreceptor subtypes to stimulate and inhibit cAMP formation, respectively(Sonnenburg, and Smith, (1988) J. Biol. Chem., 263: 6155-6160). PGE₂also stimulates calcium release and protein kinase C activity in therabbit kidney collecting tubule, most likely by binding to the EP,receptor subtype that is coupled to stimulation of phospholipase C(Hebert et al., (1990) Am. J. Physiol., 259: F318-F325). The EP₃receptor subtype is involved in inhibition of gastric acid secretion,modulation of neurotransmitter release, inhibition of sodium and waterreabsorption in the kidney tubule, potentiation of platelet aggregationat low concentrations (below 1 μM) and inhibition of plateletaggregation at higher concentrations (Tynan et al., (1984)Prostaglandins, 27: 683-696; Matthews and Jones, (1993) British J.Pharmacol., 108: 363-369).

Development of therapeutic prostaglandins requires selective action atreceptor subtypes. The murine EP₂ and EP₃ prostaglandin receptors havebeen cloned and sequenced (Honda et al., (1993) J. Biol. Chem., 268:7759-7762; Sugimoto et al., (1992) J. Biol. Chem., 267: 6463-6466). Thededuced protein sequences indicate that both are members of the Gprotein-linked receptor superfamily, having seven putativemembrane-spanning hydrophobic domains. The proteins share significantamino acid sequence similarity with other members of this familyincluding the thromboxane (TP) receptor (Hirata et al., (1991) Nature349: 617-620), rhodopsin and the adrenergic receptors. In order tocharacterize the pharmacology of the murine EP₃ receptor, the gene wastransfected into COS-7 cells which lack the EP₃ receptor and competitionbinding assays using tritiated PGE₂ were performed on the plasmamembrane fraction (Sugimoto et al., (1992) J. Biol. Chem., 267:6463-6466).

However, these results only addressed the binding of compounds to themurine receptors. There is still the need to identify compounds whichspecifically bind to the human EP₃ receptor, since the pharmacology ofrodent G-protein coupled receptors does not always match their humanhomologs (Oksenberg et al., (1992) Nature, 360: 161-163; Link et al.,(1992) Mol. Pharmacol., 42: 16-27).

SUMMARY OF THE INVENTION

One embodiment of the present invention is an isolated DNA moleculeencoding the human prostaglandin EP₃ receptor.

Preferably, this molecule has the nucleotide sequence of SEQ ID NOS: 3,5, 7 or 16.

Another embodiment of the present invention is an isolated, unique 18nucleotide DNA sequence contained within SEQ ID NOS: 3, 5, 7 or 16.

A further embodiment of the present invention are the proteins derivedfrom the aforementioned DNA sequences.

The present invention also embodies a vector containing SEQ ID NOS: 3,5, 7 or 16 operably linked to a heterologous promoter.

Another aspect of the present invention provides isolated antibodiesdirected to the human EP₃ receptor protein. Preferably, these antibodiesare polyclonal; most preferably, these antibodies are monoclonal.

A further embodiment consists of a method of screening compounds forbinding to the human EP₃ receptor by the following steps:

transfecting cells with a DNA molecule encoding a human EP₃ receptoroperably linked to a promoter;

culturing the cells to express the human EP₃ receptor;

incubating the cultured cells in the presence of a labeled compound tobe tested for binding affinity to the human EP₃ receptor; and

measuring the amount of label bound to the cells.

Preferably, the cell line is COS-7, the human EP₃ receptor is encoded bythe polynucleotide of SEQ ID NOS: 3, 5, 7, or 16, and the expressionvector is mammalian. Most preferably, the mammalian expression vector ispBC12BI. Additionally, the compound of interest may advantageously beeither radiolabeled, calorimetrically labeled or fluorimetricallylabeled. In another aspect of this preferred embodiment, prior to theincubation step, cell membranes containing the expressed human EP₃receptor are isolated.

Still another embodiment of the present invention is a method ofdetermining the ability of a compound to inhibit ligand binding to thehuman EP₃ receptor by the steps of:

transfecting cells with a DNA sequence encoding the human EP₃ receptoroperably linked to a promoter;

culturing the cells to express the human EP₃ receptor;

incubating the cultured cells in the presence of a labeled ligand havingbinding affinity for the receptor and a test compound; and

determining the level of binding of the ligand to the expressed humanEP₃ receptor, wherein a lower level of ligand binding in the presence ofthe compound indicates that the compound binds to the receptor.

Preferably, the cell line is COS-7. Advantageously, the compound may beeither radioactively, calorimetrically or fluorimetrically labeled.Alternatively, the ligand may be labeled. Most preferably, theexpression vector is pBC12BI and the ligand is PGE₂. In another aspectof this preferred embodiment, prior to the incubation step, cellmembranes containing the expressed human EP₃ receptor are isolated.

Another aspect of the present invention provides a method foridentifying compounds that are receptor agonists by the following steps:

transfecting cells with the human EP₃ receptor gene operably linked to apromoter;

transfecting cells with a DNA segment encoding cyclic AMP-responsivechloramphenicol acetyltransferase (CAT);

incubating the cells in the presence or absence of an activator ofadenylate cyclase and in the presence of a compound to be tested; and

assaying the amount of CAT produced, where a d in CAT activity indicatesthat the compound is an agonist of the receptor.

Preferably, the cell line is mammalian; most preferably, the mammaliancells are JEG-3 choriocarcinoma cells, and the activator of adenylatecyclase is forskolin.

The present invention further provides a cell line in continuous cultureexpressing the human EP₃ receptor encoded by the DNA of SEQ ID NOS: 3,5, 7 or 16. Preferably these cells are CHO cells.

FIG. 1 illustrates a competition curve of [³ H]PGE₂ binding to membranesfrom COS-7 cells transfected with the full-length hEP₃ 27 receptor. They-axis indicates the percentage of [³ H]PGE₂ specifically bound and thex-axis indicates the concentration of competitor added (-log M).

FIG. 2 illustrates a second competition curve of [³ H]PGE₂ binding tomembranes from COS-7 cells transfected with the full-length hEP₃ 27 EP₃receptor in order to more precisely define the subtype of the clonedreceptor. The y-axis indicates the percentage of [³ H]PGE₂ specificallybound and the x-axis indicates the concentration of competitor added(-log M).

FIG. 3 is a graph showing the effects of sulprostone onforskolin-stimulated chloramphenicol acetyltransferase (CAT) reportergene activity after transient transfection of JEG-3 cells with DNAencoding the reporter gene and the full-length hEP₃ 27 or the splicevariant hEP₃ 19. The sulprostone concentration (-log M) is indicated onthe x-axis and the CAT activity is indicated on the y-axis.

DETAILED DESCRIPTION OF THE INVENTION

This invention discloses the cloning, sequencing and characterization ofthe human EP₃ (hEP₃) prostaglandin receptor. The human EP₃ genesequence, fragments thereof, vectors containing this sequence or uniquefragments thereof, cells transfected with this sequence or fragmentsthereof and protein purified from these cells will be useful forstudying the pharmacology and the cellular distribution and expressionof the human EP₃ receptor.

Unexpectedly, three alternatively spliced variants of the human EP₃receptor were identified by hybridization screening of a human smallintestine cDNA library. The existence of corresponding variant mRNAs hasbeen verified by reverse transcriptase PCR. The existence of thesesplice variants was unexpected since only a single variant that resultsfrom an internal deletion has been described for the mouse receptor.Since the sequence differences occur in the carboxy-terminal regions,this may be important in receptor-effector coupling.

Fragments of the hEP₃ receptor gene consisting of at least 18consecutive nucleotides unique to hEP₃ will be useful as PCR probes forisolating other human prostaglandin receptors as well as thecorresponding receptor gene from other species. These oligonucleotideswill be useful for in situ hybridization and to probe Northern blots ofRNA isolated from various tissues by well known methods to determine thehEP₃ receptor distribution.

As specific subsets of the prostaglandin receptor family may be involvedin different cellular actions, it is important to identify the receptorsubtypes expressed by each cell. It can be appreciated that those ofordinary skill in the art could determine unique fragments of the humanEP₃ receptor and use these fragments as probes to determine cellsexpressing one of the prostaglandin receptor genes.

In addition, DNA sequences of 18 nucleotides correspond to six aminoacids. Those of ordinary skill in the art will appreciate that a sixamino acid peptide, when coupled to an immunogenic carrier protein suchas keyhole limpet hemocyanin, can be utilized as an antigen to raiseantibodies against hEP₃ receptor epitopes. Alternatively, the hEP₃ cDNAor fragments thereof can be expressed and the resulting polypeptiderecovered and used as an immunogen. Antibodies against the hEP₃ receptorprotein will allow immunohisto-chemical localization of the protein incells, tissues and body fluids, thereby providing a means foridentification of cells expressing the hEP₃ receptor subtype.

The use of a number of eukaryotic expression vectors is within the scopeof the present invention. Those of ordinary skill in the art willappreciate that once the hEP₃ receptor clone has been identified andsequenced, it can rapidly be incorporated into almost any desiredvector. In the present invention, the most preferable expression vectorsare mammalian, with the most preferable the vector being pBC12BI. Theincorporation of the alternatively spliced hEP₃ variant DNA sequencesinto expression vectors, and their subsequent transfection andexpression in mammalian cells, is also envisioned. In addition, the useof yeast, baculovirus and prokaryotic expression vectors is also withinthe scope of the present invention as is the production of the hEP₃receptor or fragments thereof in these cell types.

Assays using the expressed protein, either in whole transfected cells orin membrane preparations, will be particularly useful in theidentification of hEP₃ receptor agonists and antagonists and may aid inthe identification of pharmacologically relevant compounds. The bindingof compounds to cells expressing the receptor can then be determined.Although the preferred method of identifying receptor ligands is byradiolabeling, other methods known in the art are also within the scopeof the present invention. For instance, well known methods exist forcalorimetrically and fluorimetrically labeling compounds. One can alsomeasure functional responses in cells expressing the hEP₃ receptorprotein by using signaling systems including, but not limited to,adenylate cyclase, phosphoinositide hydrolysis, guanylate cyclase, ionfluxes and pH changes. Although JEG-3 choriocarcinoma cells were used,the use of other cell types amenable to such analysis is alsocontemplated. The response systems are either present in the host cellor can be introduced into the host cell along with the receptor.Although in the present invention the transfected cells were mammalian,any cell type able to express a transfected hEP₃ gene is within thescope of the present invention. Although transient transfection of cellshas been described, production of stable transfectants expressing thehEP₃ gene using well-known methods is also contemplated.

With the gene sequence determined, mutations can now be introduced inorder to study structure-function relationships as they relate to ligandbinding and effector system coupling. This receptor was found to haveseven putative hydrophobic membrane-spanning domains and is thusbelieved to be a member of the G protein-coupled receptor superfamily.The cloning of the hEP₃ receptor gene and analysis of the expressedprotein will help differentiate the functions of EP receptor subtypes.As a first step of isolating the hEP₃ receptor gene, PCR amplificationof human small intestine mRNA was performed as discussed in thefollowing example.

EXAMPLE 1 Cloning of a human EP₃ receptor fragment by PCR

Sense and antisense primers corresponding to the first extracellularloop and the sixth transmembrane domain, respectively, of the murine EP₃receptor were used in a PCR reaction to amplify corresponding sequencesin total RNA from human small intestine (Clontech). The sense andantisense primers used for cloning a human EP₃ receptor sequence weredesigned from nucleotides 301-326 (SEQ ID NO: 1) and 775-797 (SEQ ID NO:2), respectively, of the mouse EP₃ receptor sequence (Sugimoto et al.,(1992) J. Biol. Chem., 267: 6463-6466).

5'-GA(T/C)CCGTCG(T/G)GICGICTITG(C/T)(C/A)(C/G)ITT-3' (SEQ ID NO:1)

5'-AC(A/G)CACATIAT(A/T/G/C)CCCAT(A/T/G/C)A(A/G)(T/C)TG-3' (SEQ ID NO:2)

These primers were added to a combined reverse transcription/PCRreaction. The reverse transcription reaction contained 14 μl 10×PCRbuffer (Perkin-Elmer, Norwalk, Conn.), 3.5 μl Rnasin (BoehringerMannheim, Indianapolis, Ind.; 40 units/μl), 35 μl dNTPs (5 mM), 7 μlrandom primers (1 μg/μl), 14 μl RNA (1 μg/μl) and 7 μl AMV reversetranscriptase (Boehringer Mannheim, 25 units/Al) in a final volume of140 μl. The samples were incubated at room temperature (22° C.) for 10min, 42° C. for 1 hour, 95° C. for 5 min and placed on ice. The PCRreaction was performed using 20 μl of the reverse transcriptionreaction, 3 μ10×PCR buffer, 5 μl dimethyl sulfoxide (DMSO), 5 μl senseprimer (20 μM), 5 μl antisense primer (20 AM) and 1 μl Taq polymerase(Perkin-Elmer, 2.5 units/μl) in a final volume of 50 μl under thefollowing conditions: 94° C., 3 min; 50° C., 2 min; 72° C., 3 min; 59cycles of 94° C., 1 min; 50° C., 2 min; 72° C., 3 min; and a final cycleof 72° C., 15 min; 4° C., 12 hours.

A 15 μl sample of the PCR reaction was analyzed by electrophoresis on a100 agarose gel and stained with ethidium bromide. A single 500 basepair fragment was obtained which is in the expected size range asdeduced from the murine EP₃ receptor sequence. The band was purifiedusing Geneclean® (Bio 101, Inc., La Jolla, Calif.) and cloned intopBluescript® using a G/C tailing reaction according to themanufacturer's instructions (Boehringer Mannheim). The samples were usedto transform E. coli DH10B cells by electroporation. A positive clone(B₁) was isolated and its sequence then determined using ΔTaq-PCRsequencing according to the manufacturer's instructions (United StatesBiochemical, Cleveland, Ohio). The hEP₃ sequence exhibited significantsimilarity with the murine EP₃ receptor sequence.

To isolate a genomic clone corresponding to the B₁ cDNA, a lambdagenomic library was screened with the B₁ clone as described in thefollowing example.

EXAMPLE 2 Isolation of a genomic EP₃ receptor clone

A human genomic DNA library in λFix II (Stratagene, La

Jolla, Calif.) was screened by plaque hybridization analysis (Sambrooket al., (1989) in Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Laboratory) using a ³² p labeled B, probe as described inExample 1. Nitrocellulose filters were placed over 22 petri disheshaving a total of approximately 4.4×10⁵ recombinant clones to lift theplaque DNA. The DNA was denatured, neutralized and baked for 2 hours at80° C. The filters were prehybridized for 2 hours at 37° C. in 50%formamide, 1 M NaCl, 1% SDS, 100 μg/ml herring sperm DNA to blocknonspecific binding, then hybridized with the B₁ probe overnight at 37°C. The B₁ probe was labeled by well known methods with ³² p using a DNAnick-translation kit (Gibco/BRL) to a concentration of 5×10⁶ dpm/ml. Thefilters were washed for 1 hour at 37° C. in 2×SSC, 0.1% SDS and then for1 hour at 53° C. in 0.1×SSC, 0.1% SDS. The second wash, performed undermore stringent conditions (less salt and higher temperature) removedmore nonspecific binding. The filters were analyzed by autoradiographyand a positive clone was isolated (EP₃ λ12) after two additional plaquehybridization rounds.

DNA was prepared from plate lysates of the positive clone (EP₃ λ12)using LambdaSorb® (Promega, Madison, Wis.). The DNA was digested withEcoRI and a 13 kb insert was obtained. Further restriction analysis andSouthern blotting produced a 2.4 kb BamHI fragment that hybridized withthe ³² P-labeled B₁ clone. This 2.4 kb fragment was cloned intopBluescript® and a series of nested deletions was made with theErase-A-Base® system (Promega). Five overlapping deletion clones weresequenced by the Sequenase® deaza-7-dGTP protocol (United StatesBiochemical).

As indicated by DNA sequence analysis, an open reading frame was presentwithin this sequence which, when translated, shared greater than 80%identity with the mouse EP₃ receptor sequence. The amino terminus of thededuced amino acid sequence of the open reading frame started inapproximately the same region as that of the mouse EP₃ amino terminus,but the carboxy terminus of the human sequence ended at a pointequivalent to the putative sixth transmembrane domain of the mouse EP₃receptor. Examination of the nucleotide sequence in this region of thehuman genomic clone was consistent with an intron/exon boundarysuggesting the presence of an intron. Southern blot analysis of the fulllength EP₃ λ12 insert with a probe encoding the seventh transmembranedomain from a subsequently characterized hEP₃ receptor cDNA wasnegative, indicating that the downstream exon(s) were not present inthis genomic clone.

To isolate a longer hEP₃ receptor cDNA clone, a human small intestinecDNA library was screened with the B₁ probe as discussed in thefollowing example:

EXAMPLE 3 Isolation of EP₃ receptor cDNA clones

A human small intestine cDNA library (Clontech) in λgt10 was screenedwith the ³² p labelled B₁ PCR product as in Example 2. Approximately860,000 recombinants were screened resulting in two positive clones(hEP₃ 27 and hEP₃ 32). These clones were subcloned into the EcoRI siteof pBluescript and sequenced as described above. However, neither clonecontained a complete open reading frame. Another 460,000 recombinantswere screened under the same conditions except that the filters werewashed for 1 hour at 50° C. in 1×SSC, 0.1% SDS. Four positive cloneswere obtained (hEP₃ 1, hEP₃ 4, hEP₃ 19, hEP₃ 21), subcloned andsequenced.

Although none of the isolated cDNA clones was full length, an alignmentof the entire open reading frame could be determined by matchingoverlapping sequenced gene regions. In a third screen, 500,000recombinants were screened with a ³² P-labeled RNA probe generated fromone of the nested deletion genomic clones that ends upstream of theintron. Filters were prehybridized, then hybridized with approximately2×10⁶ dpm/ml at 60° C. in 6×SSC, 20 mM sodium phosphate, pH 7.6,1×Denhardt's solution, 1 mM dithiothreitol, and 100 μg/ml poly A RNA.The filters were washed under increasingly stringent conditions with afinal wash in 0.1×SSC, 0.1% SDS at 60° C. for 20 minutes. A singlefull-length cDNA clone was identified (EP₃ 11.2), confirming thepreviously sequenced overlapping reading frames. In addition, there wasevidence for three alternative splicing variants in the 3' regions ofthe human cDNAs since three of the clones obtained encoding this regionhad different nucleotide sequences from hEP₃ 27. These variants werecalled hEP₃ 32, hEP₃ 11.2 and hEP₃ 19 (SEQ ID NO: 3, 5, and 7,respectively). EP₃ 32 shared amino acid identity in its carboxy terminalregion with hEP₃ 27 and the mouse EP₃ α receptor, but had a completelydifferent 3' untranslated sequence. To confirm the presence of mRNAsencoding the hEP₃ receptor variants, PCR amplification of the regionspanning the divergent point was performed as described below.

EXAMPLE 4 PCR cloning of splice variants

Sense and antisense primers that span the diverged point were chosen toamplify sequences specific for each variant. The sense primer(5'-TGGCGCTGACAGTCACCT; SEQ ID NO: 9) was common to all hEP₃ receptorclones and annealed at nucleotides 774-791 of SEQ. ID NO. 16. Theantisense primers were specific to the unique 3'-untranslated regions ofthe clones as follows:

    ______________________________________                                                                      SEQ                                                                            Nucleotide #  ID                               ______________________________________                                        antisense 11                                                                          5'-GGCTGCCCTTTCTGTCCA-3'                                                                          1166-1183 10                                        antisense 19 5'-GATGTGATCCTGGCAGAA-3' 1164-1181 11                            antisense 27 5'-CAGGGAAGCAGGAATTGC-3' 1257-1274 12                            antisense 32 5'-AGGCGAAGATTGCAGTGA-3' 1356-1373 13                          ______________________________________                                    

The reverse transcription reactions contained 23 μl water, 2 μl RNaseinhibitor (50 units/μl, Boehringer Mannheim), 2 μl denatured total RNA(1 μg/μl), 6 μl 10X buffer (Boehringer Mannheim), 12 μl MgCl₂ (25 mM), 6μl dNTPs (10 mM each), 6 μl poly (dN)₆ (0.04 A₂₆₀ /μl) or 6 μl poly(dT)₁₅ (0.02 A260/μl), 0.6 μl acetylated BSA (10 mg/ml, Promega), 2.4 μlAMV reverse transcriptase (25 units/μl, Boehringer Mannheim) in a finalvolume of 60 μl. The reactions were incubated at room temperature for 10min, 42° C. for 45 min, 95° C. for 5 min and held on ice.

The PCR reactions contained 4 μl of the reverse transcription reaction,33.5 μl water, 5 μl 10× buffer (Perkin-Elmer), 3 μl MgCl₂, 2 μl dNTPs(1.25 mM each), 1 μl TMA (2.5×10⁻³ mM), 0.625 μl sense primer (25 μM),0.25 μl Taq polymerase (Perkin-Elmer, 5 units/μl) in a final volume of50 μl. There was an initial denaturation step of 94° C. for 2 min, 35cycles at 94° C. for 15 sec, 60° C. for 15 sec and 72° C. for 36 sec.Finally, there was a single 72° C., 6 min incubation. Half of the PCRreactions (25 μl) was analyzed by electrophoresis on a 1.5% agarose gelstained with ethidium bromide. These samples were run in parallel withpositive controls (plasmid DNA with template) and negative controls(plasmid DNA without template).

Fragments of the predicted size (hEP₃ 11: 409 bp; hEP₃ 19: 407 bp; hEP₃27: 500 bp; hEP₃ 32: 599 bp) were amplified with RNA from human smallintestine, heart, pancreas, and ocular ciliary muscle, confirming thepresence of all of the hEP₃ receptor variants described above.Interestingly, PCR using primers specific for variant hEP₃ 19 on humansmall intestine RNA produced a second fragment 50-100 base pairs largerthan would be expected. This could represent an additional variant thathas an insertion in the carboxy terminal region of hEP₃ 19. Since thissecond fragment was not observed in PCR of RNA isolated from othertissues, this may represent a tissue-specific variant.

The difference between the 3' splice variants of the present inventionand those of the murine receptor (Sugimoto et al., (1993) J. Biol.Chem., 268: 2712-2718) is that the human amino acid (and 3' untranslatednucleotide) sequences are all different, whereas the murine variantarises by a deletion of an 89 base pair sequence. This creates anotherreading frame downstream from this junction which extends the codingregion to a new stop codon 77 base pairs downstream from the originalstop codon. As a consequence, a 30 amino acid sequence is replaced witha 26 amino acid sequence. The variants of the present invention,however, arise by a completely different, presently unknown, mechanism.

As mentioned previously, the differences in amino acid sequence at thecarboxy termini of these variants may be significant in the regulationand functional coupling of these receptor isoforms to effectormolecules. The different carboxy-terminal regions may either interactwith different protein effectors thus initiating different enzymaticcascades, may change the efficiency of coupling to the same effectorsystem or may differentially regulate coupling by changes inphosphorylation states. The hEP₃ 19 receptor variant, which has only 6amino acids at its carboxy terminus, lacks serine, threonine andtyrosine residues, all targets for kinase-induced phosphorylation.Conversely, the carboxy terminus of the full-length hEP₃ 27 receptorcontains all of these amino acids and thus has the potential to beregulated by phosphorylation. It is important to understand thecontribution of each of these variant receptors to the overall effect ofthe ligand since this overall effect is a sum of the effects exerted bythe different receptors.

A full length functional eukaryotic expression plasmid was constructedas described in the following example.

EXAMPLE 5 Construction of a functional EP₃ expression plasmid

To construct a full length plasmid encoding the human EP₃ receptor, asense PCR primer (5'GATCCACCGCGGTGG-AATATTGCCCCCTCCCGCTGCGGCTCT-3'; SEQID NO: 14) containing adjacent BstXI and SspI restriction sites wasdesigned to have homology to a nucleotide sequence upstream of the hEP₃receptor translation start site. This primer had sequences identical tothe cDNA and genomic clones as well as an additional 5' sequence. Thisprimer was used with a corresponding antisense primer(5'-GTCCAGTGGCCCGGGACGTGGTG-3'; SEQ ID NO: 15) in a PCR reaction withthe 2.4 kb genomic clone as a template. A 700 base pair product wasobtained, purified with Geneclean (Bio 101, Inc.) and digested withBstXI. The 500 base pair fragment was isolated from a low-melting-pointagarose gel and was used in a solid phase ligation (Sambrook et al.,1989, Molecular Cloning: a Laboratory Manual, Cold Spring HarborLaboratory) with the 4.2 kb fragment isolated from the digestion ofpBluescript cDNA clone (hEP₃ 27) with BstXI. E. coli were transformedand a clone was obtained (KS/hEP₃ FulCod27) The full-length cloneKS/hEP₃ 27 Ful/Cod obtained by this procedure had 1170 bases of thecomplete open reading frame, 56 bases of 5'-untranslated sequence and504 bases of 3'-untranslated sequence (SEQ ID NO: 13). The putativetransmembrane (TM) regions were encoded by the following nucleotidesequences of SEQ ID NO: 16: TM1, 211-277; TM2, 319-398; TM3, 451-511;TM4, 571-646; TM5, 739-814; TM6, 895-964; TM7, 1039-1102. A full-lengthhEP₃ 19 clone was made by ligation of the large fragment remaining fromthe digestion of KS/hEP₃ 27 Ful/Cod with BglII and HindIII with thesmall fragment remaining from the digestion of hEP₃ 19 with the sameenzymes. A full-length hEP₃ 11 clone was made in a similar manner bydigesting the hEP₃ 11 clone with BamHI and ligating the resulting smallfragment to the large fragment remaining from the digestion of KS/hEP₃27 Ful/Cod with BamHI which had been previously modified by digestionwith SspI and cloning the sequence back into pBluescript which had beencleaved with EcoRV.

The full length clone, KS/hEP₃ 27 FulCod (SEQ ID NO: 16), was digestedwith SspI, analyzed on a 1.4% agarose gel and the 1.4 kb fragment wasthen purified. The eukaryotic expression vector pBC12BI (Cullen, (1987)Methods Enzymol., 152: 684-704) was digested with BamHI and HindIII,treated with the Klenow fragment of DNA polymerase I to produce bluntends and analyzed on a 1.4% low-melting point agarose gel. The 3.9 kbfragment was isolated and used in a solid phase ligation with the 1.4 kbSspI fragment from KS/hEP₃ FulCod/27. E. coli were transformed and aclone was obtained (pBC/hEP₃). The full-length hEP₃ 19 was inserted intothe pBC12BI vector in the same manner except that it was digested withHinfI and made blunt-ended with the Klenow fragment of DNA polymerase Iprior to SspI digestion. The full-length hEP₃ 11 was digested withHindIII and SspI and ligated into the pBC12BI vector that had beendigested with BamHI, blunt-ended with the Klenow fragment of DNApolymerase I and then digested with HindIII.

So as to perform the necessary binding assays for demonstrating theligand specificity of the protein derived from the isolated clone, thehEP₃ receptor was expressed in transfected COS-7 cells as described inthe following example.

EXAMPLE 6 Expression of the human EP₃ 27 receptor in COS-7 cells

Monolayers of COS-7 cells (70-80% confluent) were rinsed with PhosphateBuffered Saline (PBS, Ca/Mg-free) in 150×25 mm culture dishes. Ten mltransfection mix, consisting of 5 μg/ml plasmid DNA, 0.5 mg/mlDEAE-dextran in PBS, was added to each dish and cells were incubated for30 min at 37° C. Nine mls of the solution was removed from each dishfollowed by the addition of 10 ml of 100 mM chloroquine in Dulbecco'sModified Eagle Medium (DMEM)/5% fetal bovine serum (FBS). The cells wereincubated for 2.5 hr at 37° C., the solution aspirated and 10 ml of 10%dimethyl sulfoxide (DMSO) in DMEM/5% FBS was added. The cells wereincubated for 2.5 minutes at 37° C., the solution was aspirated and 30ml of DMEM/5% FBS was added. The cells were incubated at 37° C. withmedia changes at 24 and 48 hours. The media was aspirated after 72 hoursand the cells were scraped into cold TME buffer (50 mM Tris-HCl, pH 7.4,10 mM MgCl₂, 1 mM EDTA). The dishes were rinsed with cold TME buffer andthe cells combined and placed on ice.

To demonstrate the binding of hEP₃ receptor ligands to isolatedmembranes of COS-7 cells expressing the hEP³ 27 receptor, membranes wereisolated and the binding of radiolabeled ligands was assessed in thepresence of increasing concentrations of unlabeled prostaglandinreceptor agonists as described in the following example:

EXAMPLE 7 COS-7 membrane preparation and radioligand binding assay

Transfected COS-7 cells were homogenized for 30 seconds at approximately80% power with a Brinkman PT 10/35 Polytron. The homogenate wascentrifuged at 19,000 rpm for 20 minutes at 4° C. using a Sorvall SS-34rotor. The membrane pellet was resuspended in cold TME buffer (1 ml peroriginal dish), frozen in liquid nitrogen and stored at -80° C.

Membrane pellets were resuspended in ice-cold 50 mM Tris-HCl buffer atpH 7.4 by using a sonicator at the 50 watt setting. Membrane suspensions(100 μl) were then added to each assay tube to start the bindingreaction. Final concentrations were as follows: [³ H]-PGE₂, 5 nM; 60-70μg protein/tube in a total volume of 200 μl. Samples were incubated for30 minutes at room temperature. Contents were aspirated onto a presoakedice-cold Whatman GF/B filter using a Brandel Cell Harvester and washedthree times with ice-cold assay buffer. The filters were dried, placedin scintillation fluid and counted.

As shown in FIG. 1, as expected, the strongest competitor of [³ H]-PGE₂binding was PGE₂ itself. PGE₁ was also an effective competitor and isknown to bind to the hEP₃ receptor. However, PGF₂α, and PGD₂,prostaglandins specific for the FP and DP receptors, respectively,competed weakly but only at higher concentrations. The Thromboxane A₂analogue U46619 was totally ineffective in competing for [³ H]-PGE₂binding (FIG. 1).

FIG. 2 shows additional agonist competition data which further definedthe receptor subtype. MB28767, an EP₃ receptor-selective agonist, wasthe most potent competitor of [³ H]-PGE₂ binding. In fact, this compoundwas even more potent that PGE₂ itself. GR63799X and sulprostone, two EP₃receptor agonists, also competed significantly for [³ H]-PGE₂ binding.The PGE₁ and PGE₂ analogs, 11-deoxy PGE₁ and 17-phenyl PGE₂,respectively, also competed but not as well as the parent compounds.AH13205, an EP₂ -selective agonist, did not compete.

An important difference between the mouse EP₃ receptor and the human EP₃receptor is the binding affinity for various receptor agonists. Sugimotoet al. (J. Biol. Chem. (1992) 267: 6463-6466) determined that thebinding affinity of PGE₁, PGE₂, MB28767 and GR63799X for the murinereceptor were all very similar. However, the binding affinity of thesecompounds for the expressed human receptor was quite different withMB28767>PGE₂ >PGE₁ =GR63799X. This indicates that the two receptorsmight differ in their response to the same agonists and further thatthere may be important functional differences between the two receptors.

The native EP₃ receptor is coupled through a G protein to the inhibitionof adenylate cyclase, an enzyme which converts ATP to cyclic AMP (cAMP).To produce a sensitive functional assay for prostaglandin receptorbinding, the hEP₃ 27 cDNA was cotransfected with a cAMP-dependent CATreporter plasmid into human JEG-3 choriocarcinoma cells. The effects ofEP₃ receptor agonists on forskolin-induced cAMP accumulation aredescribed in the following example.

EXAMPLE 8 Drug treatment and CAT assay

Human JEG-3 cells (American Type Culture Collection, Rockville, Md.)were cultured in DMEM containing 10% FCS, 100 units/ml penicillin, 100μg/ml streptomycin. Cells were plated in 10 cm dishes 1-2 days beforetransfection. Cells were then transfected with 10 μg of a CAT reporterplasmid TESBglIICRE(+)ANHSE (provided by P. Mellon, Salk Institute, LaJolla, Calif.) containing an 18 base pair cyclic AMP responsive elementfrom the promoter of the a-subunit gene for the human glycoproteinhormones linked to the herpes simplex virus thymidine kinase promoter inturn linked to CAT (Delegeane et al., (1987) Mol. Cell. Biol., 7:3994-4002), and 10 μg hEP₃ receptor plasmid, using the calcium phosphateprecipitation technique (Graham and van der Eb, (1973) Virology, 52:456-467). After transfection, cells were maintained in DMEM/5% FCS for36-40 hours. They were then rinsed twice with DMEM. Forskolin (1 μM), adrug known to stimulate adenylate cyclase activity, and sulprostone, anEP₃ receptor agonist, were then added in 5 ml DMEM. Cells were incubatedfor 4 hours at 37° C. and harvested.

For the CAT assay, after drug incubations, cells were rinsed with coldPBS and scraped into 1 ml 40 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mMEDTA. Cells were centrifuged and lysed by 3 cycles of freeze-thaw in 200μl 250 mM Tris-HCl, pH 7.5. [³ H]-CAT assays were performed using 50 μlcytosol, 200 nCi [³ H]-chloramphenicol and 300 μM butyryl CoA (Seed andSheen, (1988) Gene, 67: 271-277). Samples were incubated for 1 hour at37° C. and reactions were stopped by addition of 200 μl mixed xylenes.Butyrylated chloramphenicol was extracted into mixed xylenes which wereback extracted twice with 200 μl 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.Radiolabeled product was measured by liquid scintillation counting usinga Packard Tri-Carb 460C at 50-52% efficiency. Increased CAT activity,indicated by transfer of butyryl groups from butyryl CoA to [³H]-chloramphenicol, was a measure of increased cAMP accumulation.

hEP₃ 27 and CRE-CAT cotransfected JEG-3 cells exhibited a biphasicresponse to increasing concentrations of sulprostone, with inhibition ofCAT activity occurring at low concentrations and a reversal occurring athigher concentrations (FIG. 3). This biphasic response was not reportedfor the mouse EP₃ receptor and further demonstrates the functionaldifferences between the murine and human EP₃ receptors.

To determine whether any functional differences existed between thefull-length hEP₃ 27 clone and the splice variants, COS-7 cells werecotransfected with the hEP₃ 19 clone. The CAT assay was performed andthe dose-response profile was compared to that of hEP₃ 27. As indicatedin FIG. 3, the dose-response curves of the two clones are clearlydifferent. The hEP₃ 27 clone is more sensitive to sulprostone than thehEP₃ 19 clone. This indicates that the clones are functionally as wellas sequentially different and exhibit different pharmacologies thatcannot be predicted from previous results.

EXAMPLE 9 Tissue distribution of EP₃ receptor gene

A multiple human tissue Northern blot (Clontech) consisting of RNAisolated from heart, brain, placenta, lung, liver, kidney and pancreaswas prehybridized in 4.4×SSPE, 44% deionized formamide, 8.8×Denhardt'ssolution, 1.75o SDS and 88 μg/ml denatured herring sperm DNA to reducebackground hybridization at 42° C. with constant rotation. Theprehybridization solution was removed and the filter was incubated infresh solution containing 1.5×10⁶ cpm/ml nick-translated hEP₃ /27/FulCodDNA (Gibco BRL, Gaithersburg, Md.) at 42° C. overnight with constantrotation. The blot was washed six times in 2×SSC, 0.5% SDS at roomtemperature for 5 minutes each with constant agitation. The blot wasthen washed in 0.1×SSC, 0.1% SDS at 50° C. for 40 minutes with onechange of solution, dried and exposed to x-ray film. The lane containingthe kidney RNA was strongly positive, while the lanes containing theheart and pancreas RNA were much weaker in intensity.

EXAMPLE 10 Production of polyclonal antibodies against human EP₃

PCR primers are used to amplify an 80 nucleotide region corresponding tothe hydrophilic amino acid segments connecting the fifth and sixthmembrane spanning domains of the human EP₃ receptor. The resulting PCRproduct is purified by agarose gel electrophoresis, cloned into anexpression plasmid such as pGEX (Pharmacia, Piscataway, NJ) and used totransform E. coli by standard procedures. The positive clones areidentified and induced to express the fusion protein, which is purifiedby well known methods.

The purified fusion protein is injected into the breast muscle ofchickens (50-100 μg/injection) with booster injections given at two weekintervals. The IgY antibodies are purified from the egg yolks by wellknown methods and their specificity determined by immunoblotting oftissue extracts.

In addition, monoclonal antibodies to the hEP₃ receptor can be preparedas discussed below.

EXAMPLE 11 Production of monoclonal antibodies against EP₃

The hEP₃ receptor-transfected COS-7 cell lysate, isolated as describedin Example 6, is centrifuged to isolate membranes. The isolatedmembranes are injected in Freund's complete adjuvant into mice. After 9booster injections over a three week period, the spleens are removed andresuspended in PBS. The resuspended spleen cells are mixed(approximately 4:1) with SP2/0 myeloma cells. Polyethylene glycol isadded to fuse the myeloma and spleen cells, and the hybridomas areselected in HAT medium. The fused cells are aliquoted to allow growth ofonly one cell in each well of a 96 well microtiter plate. Each cell isexpanded, the media removed and secreted proteins are labeled with ¹²⁵I. The labeled media from each well is used to probe a Western blot oftransfected and untransfected COS-7 cell membranes.

The desired hybridoma produces a monoclonal antibody that strongly bindsa protein band in a transfected COS-7 cell membrane lane on a Westernblot, but does not bind to any other protein in that lane or in anuntransfected COS-7 cell membrane lane (control). This method can beused to detect those cells expressing the human EP₃ receptor.

EXAMPLE 12 Production of stably-transfected cells

To produce CHO cells stably transfected with the hEP₃ gene, CHO cellsare cotransfected with 10-30 μg hEP₃ (SEQ ID NO: 13) and 1-5 μg pSV2Neocarrying the neomycin resistance gene by calcium phosphate precipitation(Graham and Van der Eb, (1973) Virology, 52: 456-467). The cells arethen subjected to selection with 600 μg/ml genetecin (G418; Gibco). Theresistant colonies are selected, expanded and screened for receptorexpression using [³ H]PGE₂ binding as described in Example 7.

While particular embodiments of the invention have been described indetail, it will be apparent to those skilled in the art that theseembodiments are exemplary, rather than limiting, and the true scope ofthe invention is that defined in the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 17                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Sense primer                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: modified.sub.-- - #base (inosine)                               (B) LOCATION: 12                                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: modified.sub.-- - #base (inosine)                               (B) LOCATION: 15                                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: modified.sub.-- - #base (inosine)                               (B) LOCATION: 18                                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: modified.sub.-- - #base (inosine)                               (B) LOCATION: 24                                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GAYCCGTCGK GNCGNCTNTG YMSNTT          - #                  - #                  26                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Antisense pr - #imer                                      - -     (ix) FEATURE:                                                                  (A) NAME/KEY: modified.sub.-- - #base (inosine)                               (B) LOCATION: 9                                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - ACRCACATNA TNCCCATNAR YTG           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1651 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 57..1226                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - TGCCCCCTCC CGCTGCGGCT CTCTGGACGC CATCCCCTCC TCACCTCGAA GC - #CAAC             56                                                                        - - ATG AAG GAG ACC CGG GGC TAC GGA GGG GAT GC - #C CCC TTC TGC ACC CGC          104                                                                       Met Lys Glu Thr Arg Gly Tyr Gly Gly Asp Al - #a Pro Phe Cys Thr Arg             1               5 - #                 10 - #                 15              - - CTC AAC CAC TCC TAC ACA GGC ATG TGG GCG CC - #C GAG CGT TCC GCC GAG          152                                                                       Leu Asn His Ser Tyr Thr Gly Met Trp Ala Pr - #o Glu Arg Ser Ala Glu                        20     - #             25     - #             30                  - - GCG CGG GGC AAC CTC ACG CGC CCT CCA GGG TC - #T GGC GAG GAT TGC GGA          200                                                                       Ala Arg Gly Asn Leu Thr Arg Pro Pro Gly Se - #r Gly Glu Asp Cys Gly                    35         - #         40         - #         45                      - - TCG GTG TCC GTG GCC TTC CCG ATC ACC ATG CT - #G CTG ACT GGT TTC GTG          248                                                                       Ser Val Ser Val Ala Phe Pro Ile Thr Met Le - #u Leu Thr Gly Phe Val                50             - #     55             - #     60                          - - GGC AAC GCA CTG GCC ATG CTG CTC GTG TCG CG - #C AGC TAC CGG CGC CGG          296                                                                       Gly Asn Ala Leu Ala Met Leu Leu Val Ser Ar - #g Ser Tyr Arg Arg Arg            65                 - # 70                 - # 75                 - # 80       - - GAG AGC AAG CGC AAG AAG TCC TTC CTG CTG TG - #C ATC GGC TGG CTG GCG          344                                                                       Glu Ser Lys Arg Lys Lys Ser Phe Leu Leu Cy - #s Ile Gly Trp Leu Ala                            85 - #                 90 - #                 95              - - CTC ACC GAC CTG GTC GGG CAG CTT CTC ACC AC - #C CCG GTC GTC ATC GTC          392                                                                       Leu Thr Asp Leu Val Gly Gln Leu Leu Thr Th - #r Pro Val Val Ile Val                       100      - #           105      - #           110                  - - GTG TAC CTG TCC AAG CAG CGT TGG GAG CAC AT - #C GAC CCG TCG GGG CGG          440                                                                       Val Tyr Leu Ser Lys Gln Arg Trp Glu His Il - #e Asp Pro Ser Gly Arg                   115          - #       120          - #       125                      - - CTC TGC ACC TTT TTC GGG CTG ACC ATG ACT GT - #T TTC GGG CTC TCC TCG          488                                                                       Leu Cys Thr Phe Phe Gly Leu Thr Met Thr Va - #l Phe Gly Leu Ser Ser               130              - #   135              - #   140                          - - TTG TTC ATC GCC AGC GCC ATG GCC GTC GAG CG - #G GCG CTG GCC ATC AGG          536                                                                       Leu Phe Ile Ala Ser Ala Met Ala Val Glu Ar - #g Ala Leu Ala Ile Arg           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - GCG CCG CAC TGG TAT GCG AGC CAC ATG AAG AC - #G CGT GCC ACC CGC        GCT      584                                                                    Ala Pro His Trp Tyr Ala Ser His Met Lys Th - #r Arg Ala Thr Arg Ala                          165  - #               170  - #               175              - - GTG CTG CTC GGC GTG TGG CTG GCC GTG CTC GC - #C TTC GCC CTG CTG CCG          632                                                                       Val Leu Leu Gly Val Trp Leu Ala Val Leu Al - #a Phe Ala Leu Leu Pro                       180      - #           185      - #           190                  - - GTG CTG GGC GTG GGC CAG TAC ACC GTC CAG TG - #G CCC GGG ACG TGG TGC          680                                                                       Val Leu Gly Val Gly Gln Tyr Thr Val Gln Tr - #p Pro Gly Thr Trp Cys                   195          - #       200          - #       205                      - - TTC ATC AGC ACC GGG CGA GGG GGC AAC GGG AC - #T AGC TCT TCG CAT AAC          728                                                                       Phe Ile Ser Thr Gly Arg Gly Gly Asn Gly Th - #r Ser Ser Ser His Asn               210              - #   215              - #   220                          - - TGG GGC AAC CTT TTC TTC GCC TCT GCC TTT GC - #C TTC CTG GGG CTC TTG          776                                                                       Trp Gly Asn Leu Phe Phe Ala Ser Ala Phe Al - #a Phe Leu Gly Leu Leu           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - GCG CTG ACA GTC ACC TTT TCC TGC AAC CTG GC - #C ACC ATT AAG GCC        CTG      824                                                                    Ala Leu Thr Val Thr Phe Ser Cys Asn Leu Al - #a Thr Ile Lys Ala Leu                          245  - #               250  - #               255              - - GTG TCC CGC TGC CGG GCC AAG GCC ACG GCA TC - #T CAG TCC AGT GCC CAG          872                                                                       Val Ser Arg Cys Arg Ala Lys Ala Thr Ala Se - #r Gln Ser Ser Ala Gln                       260      - #           265      - #           270                  - - TGG GGC CGC ATC ACG ACC GAG ACG GCC ATT CA - #G CTT ATG GGG ATC ATG          920                                                                       Trp Gly Arg Ile Thr Thr Glu Thr Ala Ile Gl - #n Leu Met Gly Ile Met                   275          - #       280          - #       285                      - - TGC GTG CTG TCG GTC TGC TGG TCT CCG CTC CT - #G ATA ATG ATG TTG AAA          968                                                                       Cys Val Leu Ser Val Cys Trp Ser Pro Leu Le - #u Ile Met Met Leu Lys               290              - #   295              - #   300                          - - ATG ATC TTC AAT CAG ACA TCA GTT GAG CAC TG - #C AAG ACA CAC ACG GAG         1016                                                                       Met Ile Phe Asn Gln Thr Ser Val Glu His Cy - #s Lys Thr His Thr Glu           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - AAG CAG AAA GAA TGC AAC TTC TTC TTA ATA GC - #T GTT CGC CTG GCT        TCA     1064                                                                    Lys Gln Lys Glu Cys Asn Phe Phe Leu Ile Al - #a Val Arg Leu Ala Ser                          325  - #               330  - #               335              - - CTG AAC CAG ATC TTG GAT CCT TGG GTT TAC CT - #G CTG TTA AGA AAG ATC         1112                                                                       Leu Asn Gln Ile Leu Asp Pro Trp Val Tyr Le - #u Leu Leu Arg Lys Ile                       340      - #           345      - #           350                  - - CTT CTT CGA AAG TTT TGC CAG ATC AGG TAC CA - #C ACA AAC AAC TAT GCA         1160                                                                       Leu Leu Arg Lys Phe Cys Gln Ile Arg Tyr Hi - #s Thr Asn Asn Tyr Ala                   355          - #       360          - #       365                      - - TCC AGC TCC ACC TCC TTA CCC TGC CAG TGT TC - #C TCA ACC TTG ATG TGG         1208                                                                       Ser Ser Ser Thr Ser Leu Pro Cys Gln Cys Se - #r Ser Thr Leu Met Trp               370              - #   375              - #   380                          - - AGC GAC CAT TTG GAA AGA TGAGAAAAAG AAGACTCAGA GA - #GCAATTCT                1256                                                                       Ser Asp His Leu Glu Arg                                                       385                 3 - #90                                                    - - GGAGGCCGGC AAGTTCAGGA TCAGGGTGCC AGCAGATTCG GTGTCTGACT GG -             #AGTGCAGT   1316                                                                 - - GGAGTGATTT CCGCTCACTG CAACCTTCAC CTCCTCCACT CACTGCAATC TT -            #CGCCTCCT   1376                                                                 - - GGGTTCAAGT GATTCTCCTG CCTCAGCCTC CCAAGTAGCT GGAATTGCAC GA -            #TGCGCCAC   1436                                                                 - - AAGCCTGGCT AATTTTTGCA TTTTTAGTAG AGATGAGTTT CACCATGTTT GC -            #CAGGCTGG   1496                                                                 - - TCTTGAACAC CTGACCTCAA GTAACCCACC CACCTTGGCC TCCCAAGAGC TG -            #GGATTACA   1556                                                                 - - GGCATGAGCC AACGTGCCTG GCCATGTTCT GATCGTTTAA TGATAGCAAC AT -            #TTAGTATT   1616                                                                 - - ATAGAGCATG AAAATGTCAA AGCGGCCCGG AATTC       - #                       - #     1651                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 390 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Lys Glu Thr Arg Gly Tyr Gly Gly Asp Al - #a Pro Phe Cys Thr        Arg                                                                               1               5 - #                 10 - #                 15             - - Leu Asn His Ser Tyr Thr Gly Met Trp Ala Pr - #o Glu Arg Ser Ala Glu                   20     - #             25     - #             30                  - - Ala Arg Gly Asn Leu Thr Arg Pro Pro Gly Se - #r Gly Glu Asp Cys Gly               35         - #         40         - #         45                      - - Ser Val Ser Val Ala Phe Pro Ile Thr Met Le - #u Leu Thr Gly Phe Val           50             - #     55             - #     60                          - - Gly Asn Ala Leu Ala Met Leu Leu Val Ser Ar - #g Ser Tyr Arg Arg Arg       65                 - # 70                 - # 75                 - # 80       - - Glu Ser Lys Arg Lys Lys Ser Phe Leu Leu Cy - #s Ile Gly Trp Leu Ala                       85 - #                 90 - #                 95              - - Leu Thr Asp Leu Val Gly Gln Leu Leu Thr Th - #r Pro Val Val Ile Val                  100      - #           105      - #           110                  - - Val Tyr Leu Ser Lys Gln Arg Trp Glu His Il - #e Asp Pro Ser Gly Arg              115          - #       120          - #       125                      - - Leu Cys Thr Phe Phe Gly Leu Thr Met Thr Va - #l Phe Gly Leu Ser Ser          130              - #   135              - #   140                          - - Leu Phe Ile Ala Ser Ala Met Ala Val Glu Ar - #g Ala Leu Ala Ile Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Pro His Trp Tyr Ala Ser His Met Lys Th - #r Arg Ala Thr Arg        Ala                                                                                             165  - #               170  - #               175             - - Val Leu Leu Gly Val Trp Leu Ala Val Leu Al - #a Phe Ala Leu Leu Pro                  180      - #           185      - #           190                  - - Val Leu Gly Val Gly Gln Tyr Thr Val Gln Tr - #p Pro Gly Thr Trp Cys              195          - #       200          - #       205                      - - Phe Ile Ser Thr Gly Arg Gly Gly Asn Gly Th - #r Ser Ser Ser His Asn          210              - #   215              - #   220                          - - Trp Gly Asn Leu Phe Phe Ala Ser Ala Phe Al - #a Phe Leu Gly Leu Leu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ala Leu Thr Val Thr Phe Ser Cys Asn Leu Al - #a Thr Ile Lys Ala        Leu                                                                                             245  - #               250  - #               255             - - Val Ser Arg Cys Arg Ala Lys Ala Thr Ala Se - #r Gln Ser Ser Ala Gln                  260      - #           265      - #           270                  - - Trp Gly Arg Ile Thr Thr Glu Thr Ala Ile Gl - #n Leu Met Gly Ile Met              275          - #       280          - #       285                      - - Cys Val Leu Ser Val Cys Trp Ser Pro Leu Le - #u Ile Met Met Leu Lys          290              - #   295              - #   300                          - - Met Ile Phe Asn Gln Thr Ser Val Glu His Cy - #s Lys Thr His Thr Glu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Lys Gln Lys Glu Cys Asn Phe Phe Leu Ile Al - #a Val Arg Leu Ala        Ser                                                                                             325  - #               330  - #               335             - - Leu Asn Gln Ile Leu Asp Pro Trp Val Tyr Le - #u Leu Leu Arg Lys Ile                  340      - #           345      - #           350                  - - Leu Leu Arg Lys Phe Cys Gln Ile Arg Tyr Hi - #s Thr Asn Asn Tyr Ala              355          - #       360          - #       365                      - - Ser Ser Ser Thr Ser Leu Pro Cys Gln Cys Se - #r Ser Thr Leu Met Trp          370              - #   375              - #   380                          - - Ser Asp His Leu Glu Arg                                                  385                 3 - #90                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1223 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 57..1220                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - TGCCCCCTCC CGCTGCGGCT CTCTGGACGC CATCCCCTCC TCACCTCGAA GC - #CAAC             56                                                                        - - ATG AAG GAG ACC CGG GGC TAC GGA GGG GAT GC - #C CCC TTC TGC ACC CGC          104                                                                       Met Lys Glu Thr Arg Gly Tyr Gly Gly Asp Al - #a Pro Phe Cys Thr Arg             1               5 - #                 10 - #                 15              - - CTC AAC CAC TCC TAC ACA GGC ATG TGG GCG CC - #C GAG CGT TCC GCC GAG          152                                                                       Leu Asn His Ser Tyr Thr Gly Met Trp Ala Pr - #o Glu Arg Ser Ala Glu                        20     - #             25     - #             30                  - - GCG CGG GGC AAC CTC ACG CGC CCT CCA GGG TC - #T GGC GAG GAT TGC GGA          200                                                                       Ala Arg Gly Asn Leu Thr Arg Pro Pro Gly Se - #r Gly Glu Asp Cys Gly                    35         - #         40         - #         45                      - - TCG GTG TCC GTG GCC TTC CCG ATC ACC ATG CT - #G CTG ACT GGT TTC GTG          248                                                                       Ser Val Ser Val Ala Phe Pro Ile Thr Met Le - #u Leu Thr Gly Phe Val                50             - #     55             - #     60                          - - GGC AAC GCA CTG GCC ATG CTG CTC GTG TCG CG - #C AGC TAC CGG CGC CGG          296                                                                       Gly Asn Ala Leu Ala Met Leu Leu Val Ser Ar - #g Ser Tyr Arg Arg Arg            65                 - # 70                 - # 75                 - # 80       - - GAG AGC AAG CGC AAG AAG TCC TTC CTG CTG TG - #C ATC GGC TGG CTG GCG          344                                                                       Glu Ser Lys Arg Lys Lys Ser Phe Leu Leu Cy - #s Ile Gly Trp Leu Ala                            85 - #                 90 - #                 95              - - CTC ACC GAC CTG GTC GGG CAG CTT CTC ACC AC - #C CCG GTC GTC ATC GTC          392                                                                       Leu Thr Asp Leu Val Gly Gln Leu Leu Thr Th - #r Pro Val Val Ile Val                       100      - #           105      - #           110                  - - GTG TAC CTG TCC AAG CAG CGT TGG GAG CAC AT - #C GAC CCG TCG GGG CGG          440                                                                       Val Tyr Leu Ser Lys Gln Arg Trp Glu His Il - #e Asp Pro Ser Gly Arg                   115          - #       120          - #       125                      - - CTC TGC ACC TTT TTC GGG CTG ACC ATG ACT GT - #T TTC GGG CTC TCC TCG          488                                                                       Leu Cys Thr Phe Phe Gly Leu Thr Met Thr Va - #l Phe Gly Leu Ser Ser               130              - #   135              - #   140                          - - TTG TTC ATC GCC AGC GCC ATG GCC GTC GAG CG - #G GCG CTG GCC ATC AGG          536                                                                       Leu Phe Ile Ala Ser Ala Met Ala Val Glu Ar - #g Ala Leu Ala Ile Arg           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - GCG CCG CAC TGG TAT GCG AGC CAC ATG AAG AC - #G CGT GCC ACC CGC        GCT      584                                                                    Ala Pro His Trp Tyr Ala Ser His Met Lys Th - #r Arg Ala Thr Arg Ala                          165  - #               170  - #               175              - - GTG CTG CTC GGC GTG TGG CTG GCC GTG CTC GC - #C TTC GCC CTG CTG CCG          632                                                                       Val Leu Leu Gly Val Trp Leu Ala Val Leu Al - #a Phe Ala Leu Leu Pro                       180      - #           185      - #           190                  - - GTG CTG GGC GTG GGC CAG TAC ACC GTC CAG TG - #G CCC GGG ACG TGG TGC          680                                                                       Val Leu Gly Val Gly Gln Tyr Thr Val Gln Tr - #p Pro Gly Thr Trp Cys                   195          - #       200          - #       205                      - - TTC ATC AGC ACC GGG CGA GGG GGC AAC GGG AC - #T AGC TCT TCG CAT AAC          728                                                                       Phe Ile Ser Thr Gly Arg Gly Gly Asn Gly Th - #r Ser Ser Ser His Asn               210              - #   215              - #   220                          - - TGG GGC AAC CTT TTC TTC GCC TCT GCC TTT GC - #C TTC CTG GGG CTC TTG          776                                                                       Trp Gly Asn Leu Phe Phe Ala Ser Ala Phe Al - #a Phe Leu Gly Leu Leu           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - GCG CTG ACA GTC ACC TTT TCC TGC AAC CTG GC - #C ACC ATT AAG GCC        CTG      824                                                                    Ala Leu Thr Val Thr Phe Ser Cys Asn Leu Al - #a Thr Ile Lys Ala Leu                          245  - #               250  - #               255              - - GTG TCC CGC TGC CGG GCC AAG GCC ACG GCA TC - #T CAG TCC AGT GCC CAG          872                                                                       Val Ser Arg Cys Arg Ala Lys Ala Thr Ala Se - #r Gln Ser Ser Ala Gln                       260      - #           265      - #           270                  - - TGG GGC CGC ATC ACG ACC GAG ACG GCC ATT CA - #G CTT ATG GGG ATC ATG          920                                                                       Trp Gly Arg Ile Thr Thr Glu Thr Ala Ile Gl - #n Leu Met Gly Ile Met                   275          - #       280          - #       285                      - - TGC GTG CTG TCG GTC TGC TGG TCT CCG CTC CT - #G ATA ATG ATG TTG AAA          968                                                                       Cys Val Leu Ser Val Cys Trp Ser Pro Leu Le - #u Ile Met Met Leu Lys               290              - #   295              - #   300                          - - ATG ATC TTC AAT CAG ACA TCA GTT GAG CAC TG - #C AAG ACA CAC ACG GAG         1016                                                                       Met Ile Phe Asn Gln Thr Ser Val Glu His Cy - #s Lys Thr His Thr Glu           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - AAG CAG AAA GAA TGC AAC TTC TTC TTA ATA GC - #T GTT CGC CTG GCT        TCA     1064                                                                    Lys Gln Lys Glu Cys Asn Phe Phe Leu Ile Al - #a Val Arg Leu Ala Ser                          325  - #               330  - #               335              - - CTG AAC CAG ATC TTG GAT CCT TGG GTT TAC CT - #G CTG TTA AGA AAG ATC         1112                                                                       Leu Asn Gln Ile Leu Asp Pro Trp Val Tyr Le - #u Leu Leu Arg Lys Ile                       340      - #           345      - #           350                  - - CTT CTT CGA AAG TTT TGC CAG GTA GCA AAT GC - #T GTC TCC AGC TGC TCT         1160                                                                       Leu Leu Arg Lys Phe Cys Gln Val Ala Asn Al - #a Val Ser Ser Cys Ser                   355          - #       360          - #       365                      - - AAT GAT GGA CAG AAA GGG CAG CCT ATC TCA TT - #A TCT AAT GAA ATA ATA         1208                                                                       Asn Asp Gly Gln Lys Gly Gln Pro Ile Ser Le - #u Ser Asn Glu Ile Ile               370              - #   375              - #   380                          - - CAG ACA GAA GCA TGA           - #                  - #                      - #  1223                                                                  Gln Thr Glu Ala                                                               385                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 388 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Met Lys Glu Thr Arg Gly Tyr Gly Gly Asp Al - #a Pro Phe Cys Thr Arg        1               5 - #                 10 - #                 15              - - Leu Asn His Ser Tyr Thr Gly Met Trp Ala Pr - #o Glu Arg Ser Ala Glu                   20     - #             25     - #             30                  - - Ala Arg Gly Asn Leu Thr Arg Pro Pro Gly Se - #r Gly Glu Asp Cys Gly               35         - #         40         - #         45                      - - Ser Val Ser Val Ala Phe Pro Ile Thr Met Le - #u Leu Thr Gly Phe Val           50             - #     55             - #     60                          - - Gly Asn Ala Leu Ala Met Leu Leu Val Ser Ar - #g Ser Tyr Arg Arg Arg       65                 - # 70                 - # 75                 - # 80       - - Glu Ser Lys Arg Lys Lys Ser Phe Leu Leu Cy - #s Ile Gly Trp Leu Ala                       85 - #                 90 - #                 95              - - Leu Thr Asp Leu Val Gly Gln Leu Leu Thr Th - #r Pro Val Val Ile Val                  100      - #           105      - #           110                  - - Val Tyr Leu Ser Lys Gln Arg Trp Glu His Il - #e Asp Pro Ser Gly Arg              115          - #       120          - #       125                      - - Leu Cys Thr Phe Phe Gly Leu Thr Met Thr Va - #l Phe Gly Leu Ser Ser          130              - #   135              - #   140                          - - Leu Phe Ile Ala Ser Ala Met Ala Val Glu Ar - #g Ala Leu Ala Ile Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Pro His Trp Tyr Ala Ser His Met Lys Th - #r Arg Ala Thr Arg        Ala                                                                                             165  - #               170  - #               175             - - Val Leu Leu Gly Val Trp Leu Ala Val Leu Al - #a Phe Ala Leu Leu Pro                  180      - #           185      - #           190                  - - Val Leu Gly Val Gly Gln Tyr Thr Val Gln Tr - #p Pro Gly Thr Trp Cys              195          - #       200          - #       205                      - - Phe Ile Ser Thr Gly Arg Gly Gly Asn Gly Th - #r Ser Ser Ser His Asn          210              - #   215              - #   220                          - - Trp Gly Asn Leu Phe Phe Ala Ser Ala Phe Al - #a Phe Leu Gly Leu Leu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ala Leu Thr Val Thr Phe Ser Cys Asn Leu Al - #a Thr Ile Lys Ala        Leu                                                                                             245  - #               250  - #               255             - - Val Ser Arg Cys Arg Ala Lys Ala Thr Ala Se - #r Gln Ser Ser Ala Gln                  260      - #           265      - #           270                  - - Trp Gly Arg Ile Thr Thr Glu Thr Ala Ile Gl - #n Leu Met Gly Ile Met              275          - #       280          - #       285                      - - Cys Val Leu Ser Val Cys Trp Ser Pro Leu Le - #u Ile Met Met Leu Lys          290              - #   295              - #   300                          - - Met Ile Phe Asn Gln Thr Ser Val Glu His Cy - #s Lys Thr His Thr Glu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Lys Gln Lys Glu Cys Asn Phe Phe Leu Ile Al - #a Val Arg Leu Ala        Ser                                                                                             325  - #               330  - #               335             - - Leu Asn Gln Ile Leu Asp Pro Trp Val Tyr Le - #u Leu Leu Arg Lys Ile                  340      - #           345      - #           350                  - - Leu Leu Arg Lys Phe Cys Gln Val Ala Asn Al - #a Val Ser Ser Cys Ser              355          - #       360          - #       365                      - - Asn Asp Gly Gln Lys Gly Gln Pro Ile Ser Le - #u Ser Asn Glu Ile Ile          370              - #   375              - #   380                          - - Gln Thr Glu Ala                                                          385                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1429 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 57..1151                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - TGCCCCCTCC CGCTGCGGCT CTCTGGACGC CATCCCCTCC TCACCTCGAA GC - #CAAC             56                                                                        - - ATG AAG GAG ACC CGG GGC TAC GGA GGG GAT GC - #C CCC TTC TGC ACC CGC          104                                                                       Met Lys Glu Thr Arg Gly Tyr Gly Gly Asp Al - #a Pro Phe Cys Thr Arg             1               5 - #                 10 - #                 15              - - CTC AAC CAC TCC TAC ACA GGC ATG TGG GCG CC - #C GAG CGT TCC GCC GAG          152                                                                       Leu Asn His Ser Tyr Thr Gly Met Trp Ala Pr - #o Glu Arg Ser Ala Glu                        20     - #             25     - #             30                  - - GCG CGG GGC AAC CTC ACG CGC CCT CCA GGG TC - #T GGC GAG GAT TGC GGA          200                                                                       Ala Arg Gly Asn Leu Thr Arg Pro Pro Gly Se - #r Gly Glu Asp Cys Gly                    35         - #         40         - #         45                      - - TCG GTG TCC GTG GCC TTC CCG ATC ACC ATG CT - #G CTG ACT GGT TTC GTG          248                                                                       Ser Val Ser Val Ala Phe Pro Ile Thr Met Le - #u Leu Thr Gly Phe Val                50             - #     55             - #     60                          - - GGC AAC GCA CTG GCC ATG CTG CTC GTG TCG CG - #C AGC TAC CGG CGC CGG          296                                                                       Gly Asn Ala Leu Ala Met Leu Leu Val Ser Ar - #g Ser Tyr Arg Arg Arg            65                 - # 70                 - # 75                 - # 80       - - GAG AGC AAG CGC AAG AAG TCC TTC CTG CTG TG - #C ATC GGC TGG CTG GCG          344                                                                       Glu Ser Lys Arg Lys Lys Ser Phe Leu Leu Cy - #s Ile Gly Trp Leu Ala                            85 - #                 90 - #                 95              - - CTC ACC GAC CTG GTC GGG CAG CTT CTC ACC AC - #C CCG GTC GTC ATC GTC          392                                                                       Leu Thr Asp Leu Val Gly Gln Leu Leu Thr Th - #r Pro Val Val Ile Val                       100      - #           105      - #           110                  - - GTG TAC CTG TCC AAG CAG CGT TGG GAG CAC AT - #C GAC CCG TCG GGG CGG          440                                                                       Val Tyr Leu Ser Lys Gln Arg Trp Glu His Il - #e Asp Pro Ser Gly Arg                   115          - #       120          - #       125                      - - CTC TGC ACC TTT TTC GGG CTG ACC ATG ACT GT - #T TTC GGG CTC TCC TCG          488                                                                       Leu Cys Thr Phe Phe Gly Leu Thr Met Thr Va - #l Phe Gly Leu Ser Ser               130              - #   135              - #   140                          - - TTG TTC ATC GCC AGC GCC ATG GCC GTC GAG CG - #G GCG CTG GCC ATC AGG          536                                                                       Leu Phe Ile Ala Ser Ala Met Ala Val Glu Ar - #g Ala Leu Ala Ile Arg           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - GCG CCG CAC TGG TAT GCG AGC CAC ATG AAG AC - #G CGT GCC ACC CGC        GCT      584                                                                    Ala Pro His Trp Tyr Ala Ser His Met Lys Th - #r Arg Ala Thr Arg Ala                          165  - #               170  - #               175              - - GTG CTG CTC GGC GTG TGG CTG GCC GTG CTC GC - #C TTC GCC CTG CTG CCG          632                                                                       Val Leu Leu Gly Val Trp Leu Ala Val Leu Al - #a Phe Ala Leu Leu Pro                       180      - #           185      - #           190                  - - GTG CTG GGC GTG GGC CAG TAC ACC GTC CAG TG - #G CCC GGG ACG TGG TGC          680                                                                       Val Leu Gly Val Gly Gln Tyr Thr Val Gln Tr - #p Pro Gly Thr Trp Cys                   195          - #       200          - #       205                      - - TTC ATC AGC ACC GGG CGA GGG GGC AAC GGG AC - #T AGC TCT TCG CAT AAC          728                                                                       Phe Ile Ser Thr Gly Arg Gly Gly Asn Gly Th - #r Ser Ser Ser His Asn               210              - #   215              - #   220                          - - TGG GGC AAC CTT TTC TTC GCC TCT GCC TTT GC - #C TTC CTG GGG CTC TTG          776                                                                       Trp Gly Asn Leu Phe Phe Ala Ser Ala Phe Al - #a Phe Leu Gly Leu Leu           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - GCG CTG ACA GTC ACC TTT TCC TGC AAC CTG GC - #C ACC ATT AAG GCC        CTG      824                                                                    Ala Leu Thr Val Thr Phe Ser Cys Asn Leu Al - #a Thr Ile Lys Ala Leu                          245  - #               250  - #               255              - - GTG TCC CGC TGC CGG GCC AAG GCC ACG GCA TC - #T CAG TCC AGT GCC CAG          872                                                                       Val Ser Arg Cys Arg Ala Lys Ala Thr Ala Se - #r Gln Ser Ser Ala Gln                       260      - #           265      - #           270                  - - TGG GGC CGC ATC ACG ACC GAG ACG GCC ATT CA - #G CTT ATG GGG ATC ATG          920                                                                       Trp Gly Arg Ile Thr Thr Glu Thr Ala Ile Gl - #n Leu Met Gly Ile Met                   275          - #       280          - #       285                      - - TGC GTG CTG TCG GTC TGC TGG TCT CCG CTC CT - #G ATA ATG ATG TTG AAA          968                                                                       Cys Val Leu Ser Val Cys Trp Ser Pro Leu Le - #u Ile Met Met Leu Lys               290              - #   295              - #   300                          - - ATG ATC TTC AAT CAG ACA TCA GTT GAG CAC TG - #C AAG ACA CAC ACG GAG         1016                                                                       Met Ile Phe Asn Gln Thr Ser Val Glu His Cy - #s Lys Thr His Thr Glu           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - AAG CAG AAA GAA TGC AAC TTC TTC TTA ATA GC - #T GTT CGC CTG GCT        TCA     1064                                                                    Lys Gln Lys Glu Cys Asn Phe Phe Leu Ile Al - #a Val Arg Leu Ala Ser                          325  - #               330  - #               335              - - CTG AAC CAG ATC TTG GAT CCT TGG GTT TAC CT - #G CTG TTA AGA AAG ATC         1112                                                                       Leu Asn Gln Ile Leu Asp Pro Trp Val Tyr Le - #u Leu Leu Arg Lys Ile                       340      - #           345      - #           350                  - - CTT CTT CGA AAG TTT TGC CAG GAG GAA TTT TG - #G GGA AAT TAAAACCTGC          1161                                                                       Leu Leu Arg Lys Phe Cys Gln Glu Glu Phe Tr - #p Gly Asn                               355          - #       360          - #       365                      - - CTTTCTGCCA GGATCACATC ACTGGAAGCT CCATGACTCT CTTTTTGTAA AA -             #GAAAAAAA   1221                                                                 - - AATCACAGAA ACACCCACCT CCCAAACTAT TCTCTTTTAC TTCTTCCCCC AA -            #GCCCACCC   1281                                                                 - - CCAAATATAA CTGTTATCCA GAAGCTGTTA TGTCCTGTTT CCATACATGT TT -            #TTGTACTT   1341                                                                 - - TTACTATATC TACATACATC AATTAAACTT ATGTCCTATT GTTTTGTGAA TT -            #TATATTTG   1401                                                                 - - CGTATACATT ATCGTATGTC CGGAATTC         - #                  - #               1429                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 365 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Met Lys Glu Thr Arg Gly Tyr Gly Gly Asp Al - #a Pro Phe Cys Thr Arg        1               5 - #                 10 - #                 15              - - Leu Asn His Ser Tyr Thr Gly Met Trp Ala Pr - #o Glu Arg Ser Ala Glu                   20     - #             25     - #             30                  - - Ala Arg Gly Asn Leu Thr Arg Pro Pro Gly Se - #r Gly Glu Asp Cys Gly               35         - #         40         - #         45                      - - Ser Val Ser Val Ala Phe Pro Ile Thr Met Le - #u Leu Thr Gly Phe Val           50             - #     55             - #     60                          - - Gly Asn Ala Leu Ala Met Leu Leu Val Ser Ar - #g Ser Tyr Arg Arg Arg       65                 - # 70                 - # 75                 - # 80       - - Glu Ser Lys Arg Lys Lys Ser Phe Leu Leu Cy - #s Ile Gly Trp Leu Ala                       85 - #                 90 - #                 95              - - Leu Thr Asp Leu Val Gly Gln Leu Leu Thr Th - #r Pro Val Val Ile Val                  100      - #           105      - #           110                  - - Val Tyr Leu Ser Lys Gln Arg Trp Glu His Il - #e Asp Pro Ser Gly Arg              115          - #       120          - #       125                      - - Leu Cys Thr Phe Phe Gly Leu Thr Met Thr Va - #l Phe Gly Leu Ser Ser          130              - #   135              - #   140                          - - Leu Phe Ile Ala Ser Ala Met Ala Val Glu Ar - #g Ala Leu Ala Ile Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Pro His Trp Tyr Ala Ser His Met Lys Th - #r Arg Ala Thr Arg        Ala                                                                                             165  - #               170  - #               175             - - Val Leu Leu Gly Val Trp Leu Ala Val Leu Al - #a Phe Ala Leu Leu Pro                  180      - #           185      - #           190                  - - Val Leu Gly Val Gly Gln Tyr Thr Val Gln Tr - #p Pro Gly Thr Trp Cys              195          - #       200          - #       205                      - - Phe Ile Ser Thr Gly Arg Gly Gly Asn Gly Th - #r Ser Ser Ser His Asn          210              - #   215              - #   220                          - - Trp Gly Asn Leu Phe Phe Ala Ser Ala Phe Al - #a Phe Leu Gly Leu Leu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ala Leu Thr Val Thr Phe Ser Cys Asn Leu Al - #a Thr Ile Lys Ala        Leu                                                                                             245  - #               250  - #               255             - - Val Ser Arg Cys Arg Ala Lys Ala Thr Ala Se - #r Gln Ser Ser Ala Gln                  260      - #           265      - #           270                  - - Trp Gly Arg Ile Thr Thr Glu Thr Ala Ile Gl - #n Leu Met Gly Ile Met              275          - #       280          - #       285                      - - Cys Val Leu Ser Val Cys Trp Ser Pro Leu Le - #u Ile Met Met Leu Lys          290              - #   295              - #   300                          - - Met Ile Phe Asn Gln Thr Ser Val Glu His Cy - #s Lys Thr His Thr Glu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Lys Gln Lys Glu Cys Asn Phe Phe Leu Ile Al - #a Val Arg Leu Ala        Ser                                                                                             325  - #               330  - #               335             - - Leu Asn Gln Ile Leu Asp Pro Trp Val Tyr Le - #u Leu Leu Arg Lys Ile                  340      - #           345      - #           350                  - - Leu Leu Arg Lys Phe Cys Gln Glu Glu Phe Tr - #p Gly Asn                          355          - #       360          - #       365                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: sense primer                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - TGGCGCTGAC AGTCACCT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: antisense 11                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - GGCTGCCCTT TCTGTCCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: antisense 19                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - GATGTGATCC TGGCAGAA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: antisense 27                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - CAGGGAAGCA GGAATTGC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: antisense 32                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - AGGCGAAGAT TGCAGTGA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - GATCCACCGC GGTGGAATAT TGCCCCCTCC CGCTGCGGCT CT    - #                      - #  42                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - GTCCAGTGGC CCGGGACGTG GTG           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1729 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -      (v) FRAGMENT TYPE: internal                                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 57..1229                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - TGCCCCCTCC CGCTGCGGCT CTCTGGACGC CATCCCCTCC TCACCTCGAA GC - #CAAC             56                                                                        - - ATG AAG GAG ACC CGG GGC TAC GGA GGG GAT GC - #C CCC TTC TGC ACC CGC          104                                                                       Met Lys Glu Thr Arg Gly Tyr Gly Gly Asp Al - #a Pro Phe Cys Thr Arg             1               5 - #                 10 - #                 15              - - CTC AAC CAC TCC TAC ACA GGC ATG TGG GCG CC - #C GAG CGT TCC GCC GAG          152                                                                       Leu Asn His Ser Tyr Thr Gly Met Trp Ala Pr - #o Glu Arg Ser Ala Glu                        20     - #             25     - #             30                  - - GCG CGG GGC AAC CTC ACG CGC CCT CCA GGG TC - #T GGC GAG GAT TGC GGA          200                                                                       Ala Arg Gly Asn Leu Thr Arg Pro Pro Gly Se - #r Gly Glu Asp Cys Gly                    35         - #         40         - #         45                      - - TCG GTG TCC GTG GCC TTC CCG ATC ACC ATG CT - #G CTG ACT GGT TTC GTG          248                                                                       Ser Val Ser Val Ala Phe Pro Ile Thr Met Le - #u Leu Thr Gly Phe Val                50             - #     55             - #     60                          - - GGC AAC GCA CTG GCC ATG CTG CTC GTG TCG CG - #C AGC TAC CGG CGC CGG          296                                                                       Gly Asn Ala Leu Ala Met Leu Leu Val Ser Ar - #g Ser Tyr Arg Arg Arg            65                 - # 70                 - # 75                 - # 80       - - GAG AGC AAG CGC AAG AAG TCC TTC CTG CTG TG - #C ATC GGC TGG CTG GCG          344                                                                       Glu Ser Lys Arg Lys Lys Ser Phe Leu Leu Cy - #s Ile Gly Trp Leu Ala                            85 - #                 90 - #                 95              - - CTC ACC GAC CTG GTC GGG CAG CTT CTC ACC AC - #C CCG GTC GTC ATC GTC          392                                                                       Leu Thr Asp Leu Val Gly Gln Leu Leu Thr Th - #r Pro Val Val Ile Val                       100      - #           105      - #           110                  - - GTG TAC CTG TCC AAG CAG CGT TGG GAG CAC AT - #C GAC CCG TCG GGG CGG          440                                                                       Val Tyr Leu Ser Lys Gln Arg Trp Glu His Il - #e Asp Pro Ser Gly Arg                   115          - #       120          - #       125                      - - CTC TGC ACC TTT TTC GGG CTG ACC ATG ACT GT - #T TTC GGG CTC TCC TCG          488                                                                       Leu Cys Thr Phe Phe Gly Leu Thr Met Thr Va - #l Phe Gly Leu Ser Ser               130              - #   135              - #   140                          - - TTG TTC ATC GCC AGC GCC ATG GCC GTC GAG CG - #G GCG CTG GCC ATC AGG          536                                                                       Leu Phe Ile Ala Ser Ala Met Ala Val Glu Ar - #g Ala Leu Ala Ile Arg           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - GCG CCG CAC TGG TAT GCG AGC CAC ATG AAG AC - #G CGT GCC ACC CGC        GCT      584                                                                    Ala Pro His Trp Tyr Ala Ser His Met Lys Th - #r Arg Ala Thr Arg Ala                          165  - #               170  - #               175              - - GTG CTG CTC GGC GTG TGG CTG GCC GTG CTC GC - #C TTC GCC CTG CTG CCG          632                                                                       Val Leu Leu Gly Val Trp Leu Ala Val Leu Al - #a Phe Ala Leu Leu Pro                       180      - #           185      - #           190                  - - GTG CTG GGC GTG GGC CAG TAC ACC GTC CAG TG - #G CCC GGG ACG TGG TGC          680                                                                       Val Leu Gly Val Gly Gln Tyr Thr Val Gln Tr - #p Pro Gly Thr Trp Cys                   195          - #       200          - #       205                      - - TTC ATC AGC ACC GGG CGA GGG GGC AAC GGG AC - #T AGC TCT TCG CAT AAC          728                                                                       Phe Ile Ser Thr Gly Arg Gly Gly Asn Gly Th - #r Ser Ser Ser His Asn               210              - #   215              - #   220                          - - TGG GGC AAC CTT TTC TTC GCC TCT GCC TTT GC - #C TTC CTG GGG CTC TTG          776                                                                       Trp Gly Asn Leu Phe Phe Ala Ser Ala Phe Al - #a Phe Leu Gly Leu Leu           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - GCG CTG ACA GTC ACC TTT TCC TGC AAC CTG GC - #C ACC ATT AAG GCC        CTG      824                                                                    Ala Leu Thr Val Thr Phe Ser Cys Asn Leu Al - #a Thr Ile Lys Ala Leu                          245  - #               250  - #               255              - - GTG TCC CGC TGC CGG GCC AAG GCC ACG GCA TC - #T CAG TCC AGT GCC CAG          872                                                                       Val Ser Arg Cys Arg Ala Lys Ala Thr Ala Se - #r Gln Ser Ser Ala Gln                       260      - #           265      - #           270                  - - TGG GGC CGC ATC ACG ACC GAG ACG GCC ATT CA - #G CTT ATG GGG ATC ATG          920                                                                       Trp Gly Arg Ile Thr Thr Glu Thr Ala Ile Gl - #n Leu Met Gly Ile Met                   275          - #       280          - #       285                      - - TGC GTG CTG TCG GTC TGC TGG TCT CCG CTC CT - #G ATA ATG ATG TTG AAA          968                                                                       Cys Val Leu Ser Val Cys Trp Ser Pro Leu Le - #u Ile Met Met Leu Lys               290              - #   295              - #   300                          - - ATG ATC TTC AAT CAG ACA TCA GTT GAG CAC TG - #C AAG ACA CAC ACG GAG         1016                                                                       Met Ile Phe Asn Gln Thr Ser Val Glu His Cy - #s Lys Thr His Thr Glu           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - AAG CAG AAA GAA TGC AAC TTC TTC TTA ATA GC - #T GTT CGC CTG GCT        TCA     1064                                                                    Lys Gln Lys Glu Cys Asn Phe Phe Leu Ile Al - #a Val Arg Leu Ala Ser                          325  - #               330  - #               335              - - CTG AAC CAG ATC TTG GAT CCT TGG GTT TAC CT - #G CTG TTA AGA AAG ATC         1112                                                                       Leu Asn Gln Ile Leu Asp Pro Trp Val Tyr Le - #u Leu Leu Arg Lys Ile                       340      - #           345      - #           350                  - - CTT CTT CGA AAG TTT TGC CAG ATC AGG TAC CA - #C ACA AAC AAC TAT GCA         1160                                                                       Leu Leu Arg Lys Phe Cys Gln Ile Arg Tyr Hi - #s Thr Asn Asn Tyr Ala                   355          - #       360          - #       365                      - - TCC AGC TCC ACC TCC TTA CCC TGC CAG TGT TC - #C TCA ACC TTG ATG TGG         1208                                                                       Ser Ser Ser Thr Ser Leu Pro Cys Gln Cys Se - #r Ser Thr Leu Met Trp               370              - #   375              - #   380                          - - AGC GAC CAT TTG GAA AGA TAATGAAAGA ACGGAGTTGG AC - #ATTTTATT                1256                                                                       Ser Asp His Leu Glu Arg                                                       385                 3 - #90                                                    - - GCAATTCCTG CTTCCCTGAA TTTGCATATT TCTTCCCACC TGAGAAGGAT AA -             #TTATATAT   1316                                                                 - - TTTAATTTGG ATTATTTCTT CATTTTTATC TTTTTATTTT AATGATTGTT TT -            #GTCAGTAA   1376                                                                 - - TACCCATGGA GATCAACTTT ATTATTATAA TCCATGCCTC TGAATATTAG AT -            #TGGTTTCT   1436                                                                 - - TGGATGGGAT TTTGATATGC ATTTAAGAAG TTGGGAAGAA TTTCACAGAT GA -            #TGATTGGA   1496                                                                 - - GGAAAAGTGA TGAAAAGAAG ACCTGTGTTC CAGGAGTTTT CTCCAACTTC AA -            #ACCTTTAC   1556                                                                 - - GTGAATCTTA ACCAAAGTGA CATCTTTACA TTTCATGATA GCTTGCTTTT GC -            #AATATGAG   1616                                                                 - - TTTGAAAAAT CAAGATAAGC TTATGATGGT GAAAAGTCAA CATATTGAGA GT -            #GATAATTC   1676                                                                 - - AATTAATAGG ATATGAACTT AACGACATAT AAAAGCAAAT GAGGGCAGGA GG - #G              1729                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 390 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Met Lys Glu Thr Arg Gly Tyr Gly Gly Asp Al - #a Pro Phe Cys Thr Arg        1               5 - #                 10 - #                 15              - - Leu Asn His Ser Tyr Thr Gly Met Trp Ala Pr - #o Asp Gly Ser Ala Glu                   20     - #             25     - #             30                  - - Ala Arg Gly Asn Leu Thr Arg Pro Pro Gly Se - #r Gly Glu Asp Cys Gly               35         - #         40         - #         45                      - - Ser Val Ser Val Ala Phe Pro Ile Thr Met Le - #u Leu Thr Gly Phe Val           50             - #     55             - #     60                          - - Gly Asn Ala Leu Ala Met Leu Leu Val Ser Ar - #g Ser Tyr Arg Arg Arg       65                 - # 70                 - # 75                 - # 80       - - Glu Ser Lys Arg Lys Lys Ser Phe Leu Leu Cy - #s Ile Gly Trp Leu Ala                       85 - #                 90 - #                 95              - - Leu Thr Asp Leu Val Gly Gln Leu Leu Thr Th - #r Pro Val Val Ile Val                  100      - #           105      - #           110                  - - Val Tyr Leu Ser Lys Gln Arg Trp Glu His Il - #e Asp Pro Ser Gly Arg              115          - #       120          - #       125                      - - Leu Cys Thr Phe Phe Gly Leu Thr Met Thr Va - #l Phe Gly Leu Ser Ser          130              - #   135              - #   140                          - - Leu Phe Ile Ala Ser Ala Met Ala Val Glu Ar - #g Ala Leu Ala Ile Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Pro His Trp Tyr Ala Ser His Met Lys Th - #r Arg Ala Thr Arg        Ala                                                                                             165  - #               170  - #               175             - - Val Leu Leu Gly Val Trp Leu Ala Val Leu Al - #a Phe Ala Leu Leu Pro                  180      - #           185      - #           190                  - - Val Leu Gly Val Gly Gln Tyr Thr Val Gln Tr - #p Pro Gly Thr Trp Cys              195          - #       200          - #       205                      - - Phe Ile Ser Thr Gly Arg Gly Gly Asn Gly Th - #r Ser Ser Ser His Asn          210              - #   215              - #   220                          - - Trp Gly Asn Leu Phe Phe Ala Ser Ala Phe Al - #a Phe Leu Gly Leu Leu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ala Leu Thr Val Thr Phe Ser Cys Asn Leu Al - #a Thr Ile Lys Ala        Leu                                                                                             245  - #               250  - #               255             - - Val Ser Arg Cys Arg Ala Lys Ala Thr Ala Se - #r Gln Ser Ser Ala Gln                  260      - #           265      - #           270                  - - Trp Gly Arg Ile Thr Thr Glu Thr Ala Ile Gl - #n Leu Met Gly Ile Met              275          - #       280          - #       285                      - - Cys Val Leu Ser Val Cys Trp Ser Pro Leu Le - #u Ile Met Met Leu Lys          290              - #   295              - #   300                          - - Met Ile Phe Asn Gln Thr Ser Val Glu His Cy - #s Lys Thr His Thr Glu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Lys Gln Lys Glu Cys Asn Phe Phe Leu Ile Al - #a Val Arg Leu Ala        Ser                                                                                             325  - #               330  - #               335             - - Leu Asn Gln Ile Leu Asp Pro Trp Val Tyr Le - #u Leu Leu Arg Lys Ile                  340      - #           345      - #           350                  - - Leu Leu Arg Lys Phe Cys Gln Ile Arg Tyr Hi - #s Thr Asn Asn Tyr Ala              355          - #       360          - #       365                      - - Ser Ser Ser Thr Ser Leu Pro Cys Gln Cys Se - #r Ser Thr Leu Met Trp          370              - #   375              - #   380                          - - Ser Asp His Leu Glu Arg                                                  385                 3 - #90                                                  __________________________________________________________________________

What is claimed is:
 1. An isolated DNA molecule encoding humanprostaglandin EP₃ receptor, said molecule having a nucleotide sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7 and SEQ ID NO:16.
 2. A vector comprising one of the sequences ofclaim 1 operably linked to a heterologous promoter.
 3. A transfectedcell expressing the human EP₃ receptor encoded by DNA selected from thegroup consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ IDNO:
 16. 4. The transfected cell of claim 3, wherein said transfectedcell is a transfected CHO cell.